WO2009128399A1

WO2009128399A1 - Oligonucleotide probe and microarray for determination of genome type of bk virus

Info

Publication number: WO2009128399A1
Application number: PCT/JP2009/057337
Authority: WO
Inventors: 平山幸一; 池谷博; 櫻田宏一
Original assignee: 東洋鋼鈑株式会社; 警察庁科学警察研究所長が代表する日本国
Priority date: 2008-04-17
Filing date: 2009-04-10
Publication date: 2009-10-22
Also published as: JPWO2009128399A1

Abstract

Disclosed is a means for rapidly and conveniently determining the genome type of a BK virus which has infected a subject and estimate the land of the subject's origin. Specifically disclosed is a set of oligonucleotide probes for determining the genome type of a BK virus which has infected a subject, which comprises oligonucleotide probes (a-1), (a-2), (a-3) and (a-4); oligonucleotide probes (b-1), (b-2), (b-3) and (b-4); oligonucleotide probes (c-1), (c-2) and (c-3); oligonucleotide probes (d-1), (d-2) and (d-3); and oligonucleotide probes (e-1), (e-2) and (e-3).

Description

Oligonucleotide probes and microarray for determining the genomic type of BK virus

The present invention relates to an oligonucleotide probe and a microarray for determining the genome type of a BK virus infecting a subject, a method for determining the genome type of a BK virus infecting a subject using these, and a subject's It relates to the method of judging the place of birth.

Serological studies conducted around the world show that the JCV virus (JCV), a type of papovavirus, is prevalent in the human population, and that most people are asymptomatically infected with JCV when they are children It was revealed. The JCV that enters the body is not completely eliminated by the immune response, and some viruses go to the kidney tissue, peripheral blood lymphocytes, and lymphoid tissues, where they persist throughout life. In order to elucidate the origin of JCV, JCV genomic DNA cloned from races around the world was analyzed, and in the process, it was revealed that the genomic type of JCV is related to race. Since then, the base sequence of the IG region (610 base pairs) of JCV genomic DNA has been determined from urine collected in various parts of the world, and the molecular phylogenetic tree is determined from the obtained base sequence by the neighbor-joining method (Neighbor-Joining method, NJ method). Was created. The phylogenetic tree has attracted attention as a novel indicator of the human population. And it has been reported that the JCV genome type detected from human urine and kidneys is used as a method for estimating the region of origin of an unidentified corpse (for example, Non-Patent Document 1).

However, it was also revealed that the above method using the JCV genome type may not be able to estimate the region of origin. Therefore, the estimation method of the birth area using BK virus (BKV) which is a kind of the same papovavirus as JCV as another marker was examined. BK virus was first detected in kidney transplant patients, and then serological studies showed that it was as prevalent in the human population as JC virus, and that most humans can be infected with BK virus as a child. It was revealed. From the sequence analysis of the VP1 gene of BK virus, it was reported that BK virus strains can be classified into four subtypes (I to IV) that correspond well with grouping based on serological survey.

Until now, in order to accurately determine the BKV genome type, a method of clarifying the entire base sequence of the VP1 gene and analyzing the molecular system has been adopted (for example, Non-Patent Document 2). However, such a conventional method has a problem that it requires a special technique for analysis and takes time. In some cases, the number of samples is very small.
JOURNAL OF CLINICAL MICROBIOLOGY, June 1995, p.1448-1451 FORENSIC SCIENCE INTERNATIONAL, 2007, p.41-46

An object of the present invention is to provide a means for quickly and easily determining the genome type of a BK virus infecting a subject and estimating the place of birth of the subject.

The present inventors succeeded in designing a set of oligonucleotide probes useful for determining the genome type of BK virus from the base sequence of BK virus genomic DNA, and completed the present invention.

That is, the present invention includes the following inventions.
(1) A set of oligonucleotide probes for determining the genome type of a BK virus that has infected a subject, including the following oligonucleotide probes:
(a-1) an oligonucleotide probe comprising a base sequence of 30 or less bases including the bases 223 to 241 of SEQ ID NO: 1, 2 or 3 or a base sequence complementary thereto (a-2) of SEQ ID NO: 4 An oligonucleotide probe comprising a base sequence of not more than 30 consecutive bases including the 223 to 241st bases or a base sequence complementary thereto (a-3) a continuous 30 comprising the 223 to 241st bases of SEQ ID NO: 5 or 6 Oligonucleotide probe comprising a nucleotide sequence of less than or equal to the base sequence or a complementary nucleotide sequence thereof (a-4) From a nucleotide sequence of not more than 30 consecutive nucleotides including the 223 to 241st bases of SEQ ID NO: 7 or a complementary nucleotide sequence thereof An oligonucleotide probe (b-1) an oligonucleotide probe comprising a base sequence of 30 bases or less including the bases 271 to 290 of SEQ ID NO: 1, 2, or 4 or a base sequence complementary thereto (b-2) An oligonucleotide probe comprising a base sequence of not more than 30 bases including the bases 271 to 290 in column No. 3 or a base sequence complementary thereto (b-3) the bases 271 to 290 of SEQ ID NO: 5 or 6 An oligonucleotide probe comprising a continuous base sequence of 30 bases or less or a base sequence complementary thereto (b-4) a continuous base sequence of 30 bases or less including the 271th to 290th bases of SEQ ID NO: 7 or complementary thereto (C-1) consisting of a base sequence of 30 or fewer consecutive bases including the bases 285 to 304 of SEQ ID NO: 1, 4, 5, 6 or 7 or a base sequence complementary thereto Oligonucleotide probe (c-2) Oligonucleotide probe (c-3) comprising a base sequence of 30 nucleotides or less or a base sequence complementary thereto, including the 285th to 304th bases of SEQ ID NO: 2 or 3 An oligonucleotide probe consisting of a base sequence of 30 or less bases including the bases 285 to 304 in column No. 7 or a base sequence complementary thereto (d-1) a sequence containing the bases 53 to 72 of SEQ ID NO: 1 An oligonucleotide probe comprising a base sequence of 30 bases or less or a base sequence complementary thereto (d-2) comprising 30 to 53 bases in a row including the 53rd to 72nd bases of SEQ ID NO: 2, 4, 5, 6 or 7 Oligonucleotide probe comprising a base sequence or a base sequence complementary thereto (d-3) Oligonucleotide comprising a base sequence of 30 bases or less including the 53rd to 72nd bases of SEQ ID NO: 3 or a base sequence complementary thereto Probe (e-1) Oligonucleotide probe (e-2) sequence consisting of a base sequence of 30 or less bases including the 81st to 100th bases of SEQ ID NO: 1, 2, 3 or 4 or a complementary base sequence thereto An oligonucleotide probe comprising a base sequence of no more than 30 bases including the 81st to 100th bases of No. 5 or 6 or a base sequence complementary thereto (e-3) comprising the 81st to 100th bases of SEQ ID NO: 7 An oligonucleotide probe comprising a continuous base sequence of 30 bases or less or a base sequence complementary thereto.
(2) A microarray for determining the genome type of a BK virus infected with a subject, wherein the set of oligonucleotide probes according to (1) is immobilized on a carrier.
(3) A microarray for estimating the place of birth of a subject, wherein the set of oligonucleotide probes according to (1) is immobilized on a carrier.
(4) The microarray according to (2) or (3), wherein the carrier has a carbon layer and a chemical modification group on the surface.
(5) A method for determining the genome type of a BK virus that has infected a subject,
Extracting DNA from a sample derived from a subject;
Amplifying a nucleic acid encoding the VP1 region of the BK virus genome using the extracted DNA as a template;
And (1) detecting the nucleic acid amplified using the set of oligonucleotide probes according to (2) to (4) or the microarray according to any one of (2) to (4).
(6) A method for estimating the place of birth of the subject based on the genome type of the BK virus determined by the method according to (5).

The present invention makes it possible to quickly and easily determine the genome type of a BK virus that has infected a subject using a small amount of sample. In addition, the present invention provides a means by which a person without advanced specialized knowledge can determine the genome type of the BK virus. Furthermore, the birthplace of the subject can be estimated based on the genome type of the BK virus.
This specification includes the contents described in the specification and claims of Japanese Patent Application No. 2008-108209, which is the basis of the priority of the present application.

BK virus is a virus that belongs to the polyomaviridae and has a human host. The set of oligonucleotide probes of the present invention detects a region (VP1 region) encoding the VP1 gene in the genome of a BK virus that has infected a subject. The VP1 region is a region of 327 base pairs (J. のうち Med. Virol. 41 (1993) 11-17) with many mutations in the BK virus genome, and the genome types in the VP1 region are type Ia, Ib-1 Type, Ib-2 type, Ic type, II type, III type, and IV type (J.rolGen. VIrol. 85 (2004) 2821-2827), reported as a region that can be classified as seven types of genomic types Has been.

In the present invention, determining the genome type of the BK virus infected with the subject includes determining the genome type of the BK virus infected with the subject who is the origin of the test sample. Estimating the person's birthplace includes estimating the birthplace of the subject who is the origin of the test sample.

The set of oligonucleotide probes of the present invention includes at least oligonucleotide probe groups (a) to (e).

The oligonucleotide probe group (a) is composed of oligonucleotide probes (a-1), (a-2), (a-3) and (a-4); the oligonucleotide probe group (b) is an oligonucleotide probe (b -1), (b-2), (b-3) and (b-4); the oligonucleotide probe group (c) comprises oligonucleotide probes (c-1), (c-2) and (c- 3); oligonucleotide probe group (d) consists of oligonucleotide probes (d-1), (d-2) and (d-3); oligonucleotide probe group (e) consists of oligonucleotide probes (e- 1), consisting of (e-2) and (e-3).

In the set of oligonucleotide probes of the present invention, each oligonucleotide probe group such as the oligonucleotide probe group (a) may contain one type of oligonucleotide probe group, or a plurality of types as long as the conditions are satisfied. An oligonucleotide probe group may be included. Similarly, in the set of oligonucleotide probes of the present invention, each oligonucleotide probe group may include one type of oligonucleotide probe as each oligonucleotide probe such as oligonucleotide probe (a-1). As long as the condition is satisfied, a plurality of types of oligonucleotide probes may be included.

Hereinafter, each oligonucleotide probe included in each oligonucleotide probe group will be described.

Oligonucleotide probe group (a):
The oligonucleotide probe (a-1) is a base sequence of 30 bases or less including or complementary to the 223 to 241st bases of SEQ ID NO: 1, 2 or 3 (common sequence: the base sequence of SEQ ID NO: 8). It is an oligonucleotide probe consisting of a base sequence. SEQ ID NOs: 1, 2 and 3 also share 37 bases downstream of the 241st base, and therefore the oligonucleotide probe (a-1) is preferably 223 to 252 of SEQ ID NOs: 1, 2 or 3. A continuous base sequence of not more than 30 bases selected from these bases, which includes the 223th to 241st bases of SEQ ID NO: 1, 2 or 3, or a base sequence complementary thereto. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 8 is preferred. The oligonucleotide probe (a-2) is an oligonucleotide probe consisting of a base sequence of 30 bases or less including the bases 223 to 241 of SEQ ID NO: 4 (base sequence of SEQ ID NO: 9) or a base sequence complementary thereto. It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 9 is preferred. Oligonucleotide probe (a-3) is a nucleotide sequence of not more than 30 consecutive nucleotides including nucleotides 223 to 241 of SEQ ID NO: 5 or 6 (common sequence: nucleotide sequence of SEQ ID NO: 10) or a complementary nucleotide sequence thereto An oligonucleotide probe consisting of SEQ ID NOS: 5 and 6 also share 18 bases upstream and 85 bases downstream of the 223 to 241st bases. Therefore, the oligonucleotide probe (a-3) is preferably 213 to A continuous base sequence of 30 bases or less selected from the 252nd base, comprising the 223th to 241st bases of SEQ ID NO: 5 or 6, or a base sequence complementary thereto. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 10 is preferred. Oligonucleotide probe (a-4) is an oligonucleotide probe comprising a base sequence of 30 bases or less including the bases 223 to 241 of SEQ ID NO: 7 (base sequence of SEQ ID NO: 11) or a base sequence complementary thereto It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 11 is preferred.

Oligonucleotide probe group (b):
The oligonucleotide probe (b-1) is a base sequence of 30 bases or less including or complementary to the 271st to 290th bases of SEQ ID NO: 1, 2 or 4 (common sequence: the base sequence of SEQ ID NO: 12). It is an oligonucleotide probe consisting of a base sequence. SEQ ID NOs: 1, 2 and 4 have 9 bases upstream and 3 bases downstream of the 271st to 290th bases, and therefore the oligonucleotide probe (b-1) is preferably SEQ ID NO: 1, 2 or A continuous base sequence of 30 bases or less selected from the 262th to 293th bases of 4, comprising the 271st to 290th bases of SEQ ID NO: 1, 2, or 4, or a complementary base sequence thereof is there. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 12 is preferred. The oligonucleotide probe (b-2) is an oligonucleotide probe comprising a base sequence of 30 bases or less including the bases 271 to 290 of SEQ ID NO: 3 (base sequence of SEQ ID NO: 13) or a complementary base sequence thereto. It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 13 is preferred. The oligonucleotide probe (b-3) is a base sequence of 30 bases or less that includes nucleotides 271 to 290 of SEQ ID NO: 5 or 6 (common sequence: base sequence of SEQ ID NO: 14) or a complementary base sequence thereof An oligonucleotide probe consisting of SEQ ID NOs: 5 and 6 are common to 65 bases upstream and 37 bases downstream of the 271st to 290th bases. Therefore, the oligonucleotide probe (b-3) is preferably selected from 261 to A continuous base sequence of 30 bases or less selected from the 300th base, which includes the 271st to 290th bases of SEQ ID NO: 5 or 6, or a base sequence complementary thereto. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 14 is preferred. The oligonucleotide probe (b-4) is an oligonucleotide probe comprising a base sequence of 30 bases or less including the bases 271 to 290 of SEQ ID NO: 7 (base sequence of SEQ ID NO: 15) or a base sequence complementary thereto. It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 15 is preferred.

Oligonucleotide probe group (c):
The oligonucleotide probe (c-1) has a base sequence of 30 bases or less that includes the 285th to 304th bases of SEQ ID NO: 1, 4, 5, 6 or 7 (common sequence: base sequence of SEQ ID NO: 16) or It is an oligonucleotide probe consisting of a complementary base sequence. SEQ ID NOs: 1, 4, 5, 6 and 7 are common to the 1 base upstream and the 23 bases downstream of the bases of 285 to 304. Therefore, the oligonucleotide probe (c-1) is preferably SEQ ID NO: A continuous base sequence of 30 bases or less selected from the 284th to 314th bases of 1, 4, 5, 6 or 7 and the 285th to 304th bases of SEQ ID NO: 1, 4, 5, 6 or 7 Or a base sequence complementary thereto. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 16 is preferred. The oligonucleotide probe (c-2) is a base sequence of 30 bases or less that includes the 285th to 304th bases of SEQ ID NO: 2 or 3 (common sequence: the base sequence of SEQ ID NO: 17) or a base sequence complementary thereto. An oligonucleotide probe consisting of SEQ ID NOs: 2 and 3 are common to 5 bases upstream and 23 bases downstream of the bases of 285 to 304. Therefore, the oligonucleotide probe (c-2) is preferably 280 to A continuous base sequence of 30 bases or less selected from the 314th base, which contains the 285th to 304th bases of SEQ ID NO: 2 or 3, or a base sequence complementary thereto. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 17 is preferred. The oligonucleotide probe (c-3) is an oligonucleotide probe comprising a base sequence of 30 bases or less that includes the 285th to 304th bases of SEQ ID NO: 7 (base sequence of SEQ ID NO: 18) or a base sequence complementary thereto. It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 18 is preferred.

Oligonucleotide probe group (d):
Oligonucleotide probe (d-1) is an oligonucleotide probe comprising a base sequence of 30 bases or less including the 53rd to 72nd bases of SEQ ID NO: 1 (base sequence of SEQ ID NO: 19) or a base sequence complementary thereto It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 19 is preferred. The oligonucleotide probe (d-2) has a base sequence of 30 bases or less including the 53rd to 72nd bases of SEQ ID NO: 2, 4, 5, 6 or 7 (common sequence: the base sequence of SEQ ID NO: 20) or It is an oligonucleotide probe consisting of a complementary base sequence. SEQ ID NOs: 2, 4, 5, 6 and 7 have 52 bases upstream and 2 bases downstream of the 53rd to 72nd bases. Therefore, the oligonucleotide probe (d-2) is preferably SEQ ID NO: A continuous base sequence of 30 or less bases selected from the 43rd to 74th bases of 2, 4, 5, 6 or 7 and the 53rd to 72nd bases of SEQ ID NO: 2, 4, 5, 6 or 7 Or a base sequence complementary thereto. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 20 is preferred. The oligonucleotide probe (d-3) is an oligonucleotide probe comprising a base sequence of 30 bases or less including the 53rd to 72nd bases of SEQ ID NO: 3 (base sequence of SEQ ID NO: 21) or a base sequence complementary thereto. It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 21 is preferred.

Oligonucleotide probe group (e):
The oligonucleotide probe (e-1) is a base sequence of 30 bases or less including or complementary to the 81st to 100th bases of SEQ ID NO: 1, 2, 3 or 4 (common sequence: the base sequence of SEQ ID NO: 22) It is an oligonucleotide probe consisting of a typical base sequence. SEQ ID NOs: 1, 2, 3 and 4 also share the 11 bases upstream and the 1 base downstream of the 81st to 100th bases. Therefore, the oligonucleotide probe (e-1) is preferably SEQ ID NO: 1, A continuous base sequence of 30 bases or less selected from 2, 3 or 4 71-101 bases, comprising 81st to 100th bases of SEQ ID NO: 1, 2, 3 or 4; or It is a complementary base sequence. An oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 22 is preferred. The oligonucleotide probe (e-2) is a base sequence of 30 bases or less that includes the 81st to 100th bases of SEQ ID NO: 5 or 6 (common sequence: the base sequence of SEQ ID NO: 23) or a base sequence complementary thereto. An oligonucleotide probe consisting of SEQ ID NOS: 5 and 6 are common to 80 bases upstream and 17 bases downstream of the 81st to 100th bases. Therefore, the oligonucleotide probe (e-2) is preferably selected from 71 to A continuous base sequence of 30 bases or less selected from the 110th base, which includes the 81st to 100th bases of SEQ ID NO: 5 or 6, or a complementary base sequence. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 23 is preferred. The oligonucleotide probe (e-3) is an oligonucleotide probe comprising a base sequence of 30 bases or less including the 81st to 100th bases of SEQ ID NO: 7 (base sequence of SEQ ID NO: 24) or a base sequence complementary thereto. It is. An oligonucleotide probe consisting of the base sequence of SEQ ID NO: 24 is preferred.

In the above description, the oligonucleotide probe having a base sequence of 30 bases or less including the Y-Zth bases of SEQ ID NO: X is, in other words, 30 bases continuous in the base sequence represented by SEQ ID NO: X The following partial sequence refers to an oligonucleotide probe containing the Y to Z bases of the SEQ ID NO.

The length of the oligonucleotide probe of the present invention is usually 30 bases or less, preferably 25 bases or less, more preferably 23 bases or less.

The oligonucleotide probe is preferably a nucleic acid, more preferably DNA. Although DNA includes both double strands and single strands, the oligonucleotide probe of the present invention is preferably single-stranded DNA.

SEQ ID NO: 1 represents the nucleotide sequence of the Ia type VP1 region, SEQ ID NO: 2 represents the nucleotide sequence of the Ib-1 type VP1 region, SEQ ID NO: 3 represents the nucleotide sequence of the Ib-2 type VP1 region, No. 4 represents the base sequence of the Ic type VP1 region, SEQ ID NO: 5 represents the base sequence of the type II VP1 region, SEQ ID NO: 6 represents the base sequence of the type III VP1 region, SEQ ID NO: 7 represents the type IV Represents the nucleotide sequence of the VP1 region. The genome sequence of each genomic type in the VP1 region can also be obtained from the gene database GenBank of NCBI (National Center for Biotechnology Information), for example.

The oligonucleotide probe of the present invention can be obtained by, for example, chemically synthesizing with a nucleic acid synthesizer. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, a nucleic acid automatic synthesizer, or the like can be used.

The oligonucleotide probe set of the present invention is preferably used in the form of a microarray by being immobilized on a carrier. As the material for the carrier, those known in the art can be used and are not particularly limited. For example, conductive materials such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and precious metals such as compounds thereof, and carbon represented by graphite and carbon fiber; single crystal silicon, amorphous Silicon materials typified by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials typified by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, Inorganic materials such as stellite and photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol , Polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and polysulfone And organic materials. The shape of the carrier is not particularly limited, but is preferably a flat plate shape.

In the present invention, a carrier having a carbon layer and a chemical modification group on the surface is preferably used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of the substrate made of the carbon layer. As the material for the substrate, those known in the art can be used, and are not particularly limited, and the same materials as those mentioned above as the carrier material can be used.

In the microarray of the present invention, a carrier having a fine flat plate structure is preferably used. The shape is not limited to a rectangle, a square, or a round shape, but a shape of 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is usually used. Since it is easy to produce a carrier having a fine flat plate structure, it is preferable to use a substrate made of a silicon material or a resin material. In particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon has a slight change in the orientation of the crystal axis in some parts (sometimes referred to as a mosaic crystal), or includes atomic scale disturbances (lattice defects) Are also included.

In the present invention, the carbon layer formed on the substrate is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (eg, diamond-like carbon), amorphous carbon, carbon-based material (eg, graphite, fullerene) , Carbon nanotubes), a mixture thereof, or a laminate of them is preferably used. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, the soft diamond is a generic term for an incomplete diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The carbon layer has excellent chemical stability, can withstand subsequent reactions in the introduction of chemical modification groups and binding to the analyte, and the binding is flexible because of the electrostatic binding to the analyte. It is advantageous in that it has the property of being transparent, it is transparent to the detection system UV because there is no UV absorption, and it can be energized during electroblotting. Further, it is advantageous in that nonspecific adsorption is small in the binding reaction with the analyte. As described above, a carrier whose substrate itself is made of a carbon layer may be used.

In the present invention, the carbon layer can be formed by a known method. For example, microwave plasma CVD (Chemical vapor deposition) method, ECRCVD (Electric cyclotron resonance chemical vapor deposition) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionization deposition method, arc Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

In the high-frequency plasma CVD method, a raw material gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency to synthesize a carbon layer on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The carbon layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of the remaining methane gas.

In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

When the carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually a monomolecular layer to about 100 μm. If it is too thin, the surface of the base substrate may be locally exposed, and conversely thicker. In this case, productivity is deteriorated, so that the thickness is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

The oligonucleotide probe can be firmly immobilized on the carrier by introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.

An amino group can be introduced, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiation with ultraviolet rays in chlorine gas and further irradiated with ultraviolet rays in ammonia gas. Alternatively, the reaction can be carried out by reacting with a chlorinated carbon layer in a polyvalent amine gas such as methylenediamine or ethylenediamine.

The introduction of the carboxyl group can be carried out, for example, by reacting an appropriate compound with the carbon layer aminated as described above. As a compound used for introducing a carboxyl group, for example, represented by the formula: XR ¹ -COOH (wherein X represents a halogen atom, R ¹ represents a divalent hydrocarbon group having 10 to 12 carbon atoms) Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R ² -COOH ( Wherein R ² represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; poly Polyvalent carboxylic acids such as acrylic acid, polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; formula: R ³ —CO—R ⁴ —COOH (where R ³ is a hydrogen atom or 2 having 1 to 12 carbon atoms) Valent hydrocarbon group, R ⁴ represents a divalent hydrocarbon group having 1 to 12 carbon atoms) Keto acid or aldehyde acid; formula: X—OC—R ⁵ —COOH (wherein X represents a halogen atom, R ⁵ represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms). Monohalides of the indicated dicarboxylic acids such as succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

The introduction of the epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

The introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

The introduction of the hydroxyl group can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

The active ester group means an ester group having a highly acidic electron-withdrawing group on the alcohol side of the ester group and activating a nucleophilic reaction, that is, an ester group having a high reaction activity. The ester group has an electron-withdrawing group on the alcohol side of the ester group and is activated more than the alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as. More specifically, examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group and the like. In particular, an N-hydroxysuccinimide group is preferably used.

The introduction of the active ester group is performed, for example, by converting the carboxyl group introduced as described above into a dehydrating condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N- It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

The oligonucleotide probe of the present invention is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted on a carrier by a spotter device or the like. By doing so, a microarray in which oligonucleotide probes are immobilized on a carrier can be produced. Alternatively, the spotting solution may be spotted manually with a micropipette.

After spotting, it is preferable to carry out incubation in order to advance the reaction in which the oligonucleotide probe binds to the carrier. Incubation is usually performed at a temperature of −20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed in a high humidity atmosphere, for example, at a humidity of 50 to 90%. Following the incubation, it is preferable to perform washing with a washing solution (for example, 50 mM TBS / 0.05% Tween 20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) to remove DNA not bound to the carrier. .

The set of oligonucleotide probes of the present invention may be immobilized on the same carrier, or may be immobilized on different carriers, but at least the oligonucleotide probes contained in the same oligonucleotide probe group are the same. It is preferable to be immobilized on the carrier. A plurality of types of oligonucleotide probes of the present invention may be immobilized on a carrier as long as each condition is satisfied.

The present invention also relates to a method for determining the genome type of a BK virus that has infected a subject using the set of oligonucleotide probes or the microarray. The determination method of the present invention includes:
Extracting DNA from a sample derived from a subject;
Amplifying a nucleic acid encoding the VP1 region of the BK virus genome using the extracted DNA as a template;
Using the set of oligonucleotide probes or microarray of the present invention to detect the amplified nucleic acid.

The subject is usually a human, and the subject-derived sample is not particularly limited as long as it can contain a BK virus. For example, blood-related samples (blood, serum, plasma, etc.), body fluids such as lymph, sweat, tears, saliva, urine, feces, ascites and cerebrospinal fluid, and cells, tissues or organs (eg heart, pancreas, liver, ovary) , Lungs, brain, bone marrow, lymph nodes, kidneys, etc.) and the like. Preferably, kidney crushed material and extract are used. The sample derived from the subject may not be collected directly from the human body, and may be, for example, a sample collected from urine spots or blood stains.

First, DNA is extracted from a sample collected from the subject. The extraction means is not particularly limited. For example, a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB or the like can be used.

Next, an amplification reaction is performed using the obtained DNA as a template to amplify a nucleic acid encoding the VP1 region of the BK virus, preferably DNA. As the amplification reaction, polymerase chain reaction (PCR), LAMP (Loop-MediatedMediIsothermal Amplification), ICAN (Isothermal and Chimerichiprimer-initiated Amplification of Nucleic acids) method or the like can be applied. In the amplification reaction, it is desirable to add a label so that the amplified region can be identified. At this time, the method for labeling the amplified nucleic acid is not particularly limited. For example, a method in which a primer used in the amplification reaction is labeled in advance may be used, or a labeled nucleotide is used as a substrate in the amplification reaction. You may use the method to do. The labeling substance is not particularly limited, and radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

This reaction system also includes buffers necessary for nucleic acid amplification and labeling, heat-resistant DNA polymerase, primers specific to the VP1 region of BK virus, and labeled nucleotide triphosphates (specifically, nucleotide triphosphates to which a fluorescent label or the like is added). It is a reaction system containing phosphoric acid), nucleotide triphosphate, magnesium chloride and the like.

The primer used in the amplification reaction is not particularly limited as long as it can specifically amplify the VP1 region in the BK virus genome, and can be appropriately designed by those skilled in the art. For example, a primer set consisting of a primer consisting of the 1st to 20th bases and a primer consisting of the 327th to 308th bases in the VP1 region of the BK virus genome can be mentioned. In particular,
Primer 1: 5′-CAAGTGCCAAAACTACTAAT-3 ′ (SEQ ID NO: 25) and Primer 2: 5′-TGCATGAAGGTTAAGCATGC-3 ′ (SEQ ID NO: 26)
A set of primers consisting of

The amount of nucleic acid hybridized to each oligonucleotide probe can be measured, for example, by detecting a label, by carrying out a hybridization reaction between the amplified nucleic acid obtained as described above and the oligonucleotide probe of the present invention. For example, when a fluorescent label is used, the signal intensity from the label can be quantified by detecting the fluorescent signal using a fluorescent scanner and analyzing the detected signal with image analysis software. The amplified nucleic acid hybridized with the oligonucleotide probe can also be quantified by creating a calibration curve using a sample containing a known amount of DNA, for example. The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed.For example, after hybridization at 50 ° C. for 16 hours, 2 × SSC / 0.2% SDS, 25 This refers to the conditions for washing at 5 ° C for 10 minutes and 2 x SSC at 25 ° C for 5 minutes.

In the above hybridization reaction, it is preferable to use a microarray in which the set of oligonucleotide probes of the present invention is immobilized on a carrier, and apply the amplified nucleic acid to the microarray.

The method for determining the genome type of the BK virus of the present invention is carried out by comparing the amount of the amplified nucleic acid hybridized to each oligonucleotide probe within each oligonucleotide probe group. Specifically, in the oligonucleotide probe group, the amount of amplified nucleic acid hybridized to each oligonucleotide probe (for example, corresponding to the signal intensity derived from the label) is ranked. Since the order in each oligonucleotide probe group may or may not be characteristic of a particular genomic type, which genomic type has a characteristic signal for a sample from a subject By determining, the genome type of the BK virus that has infected the subject can be determined.

When the VP1 region is amplified and the amplified nucleic acid is hybridized to the set of oligonucleotide probes of the present invention for the BK virus genomic type currently known, the amount of amplified nucleic acid hybridized to each oligonucleotide probe is Each probe group has the following characteristics. That is, the ratio of the amount of amplified nucleic acid that hybridizes to each oligonucleotide probe is detected as the signal intensity ratio.

For example, when the BK virus infecting the subject is type Ia, the amount of amplified nucleic acid hybridized to the oligonucleotide probe (a-1) when viewed with respect to the oligonucleotide probe group (a) is More than the amount of amplified nucleic acid hybridized to each of the probes (a-2), (a-3) and (a-4) is detected.

In addition, when the BK virus infecting the subject is type IV, the amount of amplified nucleic acid hybridized to the oligonucleotide probe (c-1) when the oligonucleotide probe group (c) is viewed is When the amount of amplified nucleic acid hybridized to each of probes (c-2) and (c-3) is detected more than the amount of amplified nucleic acid hybridized to oligonucleotide probe (c-3) There may be cases where more than the amount of amplified nucleic acid hybridized to each of (c-1) and (c-2) is detected.

Some of the signal intensity ratios shown in Table 1 above can be seen overlapping with different genotypes, so the genotype is determined by combining the signal intensity ratios in the oligonucleotide probe groups (a) to (e). It is preferable to do. However, some of the signal intensity ratios can be found only in a specific genotype, and therefore, if the signal intensity ratio is detected, the genotype can be specified without looking at other signal intensity ratios. Therefore, among the signal intensity ratios shown in Table 1, the signal intensity ratios effective for genotype determination are shown in Table 2 below.

For example, the genotype can be specified as follows.
Amplification in which the amount of amplified nucleic acid hybridized to oligonucleotide probe (d-1) was hybridized to oligonucleotide probes (d-2) and (d-3) when viewed with respect to oligonucleotide probe group (d) If the amount is greater than the amount of nucleic acid, the BK virus that has infected the subject can be determined to be type Ia without examining other oligonucleotide probe groups.

Further, when the oligonucleotide probe group (b) is viewed, the amount of the amplified nucleic acid hybridized to the oligonucleotide probe (b-1) is the same as that of the oligonucleotide probes (b-2), (b-3) and (b- More than the amount of amplified nucleic acid hybridized to 4) and the amount of amplified nucleic acid hybridized to oligonucleotide probe (c-2) when viewed for oligonucleotide probe group (c) was -1) and the amount of amplified nucleic acid hybridized to (c-3), the BK virus that infects the subject is of type Ib-1 without examining other oligonucleotide probe groups. It can be determined that there is.

When the oligonucleotide probe group (b) is viewed, the amount of the amplified nucleic acid hybridized to the oligonucleotide probe (b-2) is the oligonucleotide probe (b-1), (b-3) and (b-4). If the amount of amplified nucleic acid hybridized to is greater than the amount of amplified nucleic acid, it can be determined that the BK virus infecting the subject is type Ib-2 without examining other oligonucleotide probe groups. When the oligonucleotide probe group (d) was viewed, the amount of amplified nucleic acid hybridized to the oligonucleotide probe (d-3) was hybridized to the oligonucleotide probes (d-1) and (d-2). Even when the amount of the amplified nucleic acid is larger, it is possible to determine that the BK virus infecting the subject is type Ib-2 without examining other oligonucleotide probe groups.

When the oligonucleotide probe group (b) is viewed, the amount of the amplified nucleic acid hybridized to the oligonucleotide probe (b-3) is the oligonucleotide probe (b-1), (b-2) and (b-4). If the amount of amplified nucleic acid hybridized to is greater than the amount of amplified nucleic acid, it can be determined that the BK virus infecting the subject is type II or type III without examining other oligonucleotide probe groups. When the oligonucleotide probe group (e) was viewed, the amount of amplified nucleic acid hybridized to the oligonucleotide probe (e-2) hybridized to the oligonucleotide probes (e-1) and (e-3) Even when the amount of the amplified nucleic acid is larger, it is possible to determine that the BK virus infecting the subject is type II or type III without examining other oligonucleotide probe groups.

Since BK virus is prevalent in the human population and each genome type has a unique distribution range around the world, by determining the genome type of the BK virus that has infected the subject, You can estimate your hometown. Therefore, the present invention also relates to a method for estimating the place of birth of a subject based on the BK virus genome type determined by the above method using the set of oligonucleotide probes or microarray of the present invention.

The distribution range of each genome type is described in detail in Microbes Infect. 9 (2) (2007) 204-13. The specific distribution of the seven main genome types is that type Ia is distributed in Africa, type Ib-1 is distributed in Southeast Asia, type Ib-2 is distributed in Europe, Type Ic is distributed in Far East Asia centering on Japan. Type II and Type III have no regional difference, but type IV subgroups are distributed in continental Asia (J Mol Evol. 65 (1) 2007 103-11.).

Example 1 Production of Support A two-layer DLC layer was formed on a 3 mm square silicon substrate using the ionized vapor deposition method under the following conditions.

An amino group was introduced onto a silicon substrate having a DLC layer on the obtained surface using ammonia plasma under the following conditions.

It was immersed in a 1-methyl-2-pyrrolidone solution containing 140 mM succinic anhydride and 0.1 M sodium borate for 30 minutes to introduce carboxyl groups. Silicon substrate surface is activated by immersing in a solution containing 0.1M potassium phosphate buffer, 0.1M アミノ 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide, 20mM N-hydroxysuccinimide for 30 minutes A carrier having a DLC layer and an N-hydroxysuccinimide group as a chemical modification group was obtained.

Example 2 Preparation of microarray 17 oligonucleotides consisting of 19 or 20 bases based on 7 genomes of BK virus (SEQ ID NOs: 1 to 7) and modified at the 5 'end with an amino group A probe was synthesized. The sequences of the oligonucleotide probes (hereinafter referred to as probes) are shown in Table 5 below. Regarding the probes described in Table 5, the corresponding oligonucleotide probe group of the present invention and each oligonucleotide probe are described in the column described as probe.

The 17 types of probes shown in Table 5 were dissolved in a spot solution (Sol. 6) so as to have a concentration of 10 pmol / μl, and a spotter SPBIO (manufactured by Hitachi Software Co., Ltd.) was used on the carrier prepared in Example 1. Spotted. After baking at 80 ° C. for 1 hour, the plate was washed with 2 × SSC / 0.2% SDS solution (room temperature) for 15 minutes, and further washed with 2 × SSC / 0.2% SDS solution (95 ° C.) for 5 minutes. The microarray in which 17 kinds of probes were immobilized on the carrier was obtained by rinsing with ultrapure water three times, removing water with a centrifuge and drying.

Example 3 Analysis of signals characteristic of BK virus genome type BK virus genome type (Ia type, Ib-1, Ib-2 type, Ic type, II type, III type, IV type) ) Has already been identified by the conventional method (method of determining the base sequence of the amplified VP1 region and determining the genome type by phylogenetic analysis, Forensic Sci. Int. 173 (1) (2007) 54-60) For the samples derived from the subjects (samples 1 to 11), the VP1 region amplified using the microarray prepared in Example 2 was detected.

A sample (a crushed kidney tissue) was collected, and DNA was extracted using QiaAmp DNA (manufactured by QIAGEN). Using this DNA as a template DNA, a PCR reaction solution was prepared with the composition shown in Table 6 so as to amplify the VP1 region (327 bp) of the BK virus.

The primer sequences used are as follows.
Primer 1: 5'-CAAGTGCCAAAACTACTAAT-3 '(SEQ ID NO: 25)
Primer 2: 5′-TGCATGAAGGTTAAGCATGC-3 ′ (SEQ ID NO: 26)
A PCR reaction was performed on the prepared PCR reaction solution using GeneAmp PCR system 9700 (ABI) at the following temperature cycle, and the VP1 region of the BK virus genome was amplified for each sample.

At this time, Cy5-dCTP was fluorescently labeled by incorporating it into the PCR product.
2 μL PCR product and 1 μL 3 × SSC / 0.3% SDS were mixed, dropped onto the microarray prepared in Example 2, and covered. Placed in a watered tapper and incubated at 45 ° C. for 1 hour. Washing was performed twice with 2 × SSC / 0.2% SDS and twice with 2 × SSC at room temperature.

The washed microarray was centrifuged at 1000 rpm and dried. Subsequently, scanning was performed with FLA 8000 (manufactured by Fujifilm), and the fluorescence intensity of each spot was digitized from the fluorescence image using Array Gauge (manufactured by Fujifilm).

The signal intensity values obtained for each sample are shown in Table 8 below. The numerical value corresponding to the signal in the probe for which the strongest signal was obtained in each probe group was shaded, and the genotype was determined based on Table 2 above. The judgment results are listed in the bottom line.

From the results of Samples 1, 2, 5 and 7, the signal intensity in the probe group (e) is e-3> e-1, e-2, so that it can be determined as type IV. Moreover, it can also be determined as type IV because the signal intensity in the probe group (b) is b-4> b-1, b-2, b-3. Looking at the results of samples 3 and 6, since the signal intensity in the probe group (b) is b-2> b-1, b-3, b-4, it can be determined as type Ib-2, and the probe group ( Since the signal intensity in d) is d-3> d-1, d-2, it can be determined as type Ib-2. Looking at the results of samples 4 and 10, in the probe group (b), the signal intensity is b-1> b-2, b-3, b-4, and in the probe group (c), the signal intensity is c- Since 2> c-1 and c-3, it can be determined as type Ib-1. Looking at the result of Sample 8, the signal intensity in the probe group (a) is a-2> a-1, a-3, a-4, and therefore it can be determined as the Ic type. Looking at the results of sample 9, since the signal intensity in probe group (b) is b-3> b-1, b-2, b-4, it can be determined as type II or type III, and probe group (e ), The signal intensity is e-2> e-1 and e-3, so that it can be determined as type II or type III. When the result of the sample 11 is seen, it can be determined as the type Ia because the signal intensity in the probe group (d) is d-1> d-2, d-3.

The genotype result determined using the microarray was consistent with the genotype result determined by the conventional method (method of determining the base sequence of the amplified VP1 region and determining the genome type by phylogenetic analysis). .

From the above, it is shown that the method of the present invention can easily and accurately determine the genome type of the BK virus possessed by the subject from the sample derived from the subject, and can estimate the birthplace of the subject. It was.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A set of oligonucleotide probes for determining the genomic type of a BK virus that has infected a subject, including the following oligonucleotide probes:
(a-1) an oligonucleotide probe comprising a base sequence of 30 or less bases including the bases 223 to 241 of SEQ ID NO: 1, 2 or 3 or a base sequence complementary thereto (a-2) of SEQ ID NO: 4 An oligonucleotide probe comprising a base sequence of not more than 30 consecutive bases including the 223 to 241st bases or a base sequence complementary thereto (a-3) a continuous 30 comprising the 223 to 241st bases of SEQ ID NO: 5 or 6 Oligonucleotide probe comprising a nucleotide sequence of less than or equal to the base sequence or a complementary nucleotide sequence thereof (a-4) From a nucleotide sequence of not more than 30 consecutive nucleotides including the 223 to 241st bases of SEQ ID NO: 7 or a complementary nucleotide sequence thereof An oligonucleotide probe (b-1) an oligonucleotide probe comprising a base sequence of 30 bases or less including the bases 271 to 290 of SEQ ID NO: 1, 2, or 4 or a base sequence complementary thereto (b-2) An oligonucleotide probe comprising a base sequence of not more than 30 bases including the bases 271 to 290 in column No. 3 or a base sequence complementary thereto (b-3) the bases 271 to 290 of SEQ ID NO: 5 or 6 An oligonucleotide probe comprising a continuous base sequence of 30 bases or less or a base sequence complementary thereto (b-4) a continuous base sequence of 30 bases or less including the 271th to 290th bases of SEQ ID NO: 7 or complementary thereto (C-1) consisting of a base sequence of 30 or fewer consecutive bases including the bases 285 to 304 of SEQ ID NO: 1, 4, 5, 6 or 7 or a base sequence complementary thereto Oligonucleotide probe (c-2) Oligonucleotide probe (c-3) comprising a base sequence of 30 nucleotides or less or a base sequence complementary thereto, including the 285th to 304th bases of SEQ ID NO: 2 or 3 An oligonucleotide probe consisting of a base sequence of 30 or less bases including the bases 285 to 304 in column No. 7 or a base sequence complementary thereto (d-1) a sequence containing the bases 53 to 72 of SEQ ID NO: 1 An oligonucleotide probe comprising a base sequence of 30 bases or less or a base sequence complementary thereto (d-2) comprising 30 to 53 bases in a row including the 53rd to 72nd bases of SEQ ID NO: 2, 4, 5, 6 or 7 Oligonucleotide probe comprising a base sequence or a base sequence complementary thereto (d-3) Oligonucleotide comprising a base sequence of 30 bases or less including the 53rd to 72nd bases of SEQ ID NO: 3 or a base sequence complementary thereto Probe (e-1) Oligonucleotide probe (e-2) sequence consisting of a base sequence of 30 or less bases including the 81st to 100th bases of SEQ ID NO: 1, 2, 3 or 4 or a complementary base sequence thereto An oligonucleotide probe comprising a base sequence of no more than 30 bases including the 81st to 100th bases of No. 5 or 6 or a base sequence complementary thereto (e-3) comprising the 81st to 100th bases of SEQ ID NO: 7 An oligonucleotide probe comprising a continuous base sequence of 30 bases or less or a base sequence complementary thereto.
A microarray for determining the genome type of a BK virus that has infected a subject, wherein the set of oligonucleotide probes according to claim 1 is immobilized on a carrier.
A microarray for estimating the place of birth of a subject, wherein the set of oligonucleotide probes according to claim 1 is immobilized on a carrier.
The microarray according to claim 2 or 3, wherein the carrier has a carbon layer and a chemical modification group on the surface.
A method for determining the genome type of a BK virus that has infected a subject,
Extracting DNA from a sample derived from a subject;
Amplifying a nucleic acid encoding the VP1 region of the BK virus genome using the extracted DNA as a template;
Detecting the nucleic acid amplified using the set of oligonucleotide probes of claim 1 or the microarray of any one of claims 2-4.
A method for estimating the place of birth of a subject based on the genome type of the BK virus determined by the method according to claim 5.